Nicotinamide riboside kinase compositions and methods for using the same

ABSTRACT

The present invention relates to isolated nicotinamide riboside kinase (Nrk) nucleic acid sequences, vectors and cultured cells containing the same, and Nrk polypeptides encoded thereby. Methods for identifying individuals or tumors susceptible to nicotinamide riboside-related prodrug treatment and methods for treating cancer by administering an Nrk nucleic acid sequence or polypeptide in combination with a nicotinamide riboside-related prodrug are also provided. The present invention further provides screening methods for isolating a nicotinamide riboside-related prodrug and identifying a natural source of nicotinamide riboside.

INTRODUCTION

This application is a continuation of U.S. patent application Ser. No.11/912,400 filed Nov. 20, 2007 now U.S. Pat. No. 8,197,807, which is theNational Stage of International Application No. PCT/US2006/015495 filedApr. 20, 2006, which claims benefit of priority to U.S. patentapplication Ser. No. 11/113,701 filed Apr. 25, 2005, the teachings ofwhich are incorporated herein by reference in their entireties.

This invention was made with government support under grant numberCA77738 awarded by the National Cancer Institute. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Nicotinic acid and nicotinamide, collectively niacins, are the vitaminforms of nicotinamide adenine dinucleotide (NAD+). Eukaryotes cansynthesize NAD+ de novo via the kynurenine pathway from tryptophan(Krehl, et al. (1945) Science 101:489-490; Schutz and Feigelson (1972)J. Biol. Chem. 247:5327-5332) and niacin supplementation prevents thepellagra that can occur in populations with a tryptophan-poor diet. Itis well-established that nicotinic acid is phosphoribosylated tonicotinic acid mononucleotide (NaMN), which is then adenylylated to formnicotinic acid adenine dinucleotide (NaAD), which in turn is amidated toform NAD+ (Preiss and Handler (1958) J. Biol. Chem. 233:488-492; Preissand Handler (1958b) J. Biol. Chem. 233:493-50).

NAD+ was initially characterized as a co-enzyme for oxidoreductases.Though conversions between NAD+, NADH, NADP and NADPH would not beaccompanied by a loss of total co-enzyme, it was discovered that NAD+ isalso turned over in cells for unknown purposes (Maayan (1964) Nature204:1169-1170). Sirtuin enzymes such as Sir2 of S. cerevisiae and itshomologs deacetylate lysine residues with consumption of an equivalentof NAD+ and this activity is required for Sir2 function as atranscriptional silencer (Imai, et al. (2000) Cold Spring Harb. Symp.Quant. Biol. 65:297-302). NAD⁺-dependent deacetylation reactions arerequired not only for alterations in gene expression but also forrepression of ribosomal DNA recombination and extension of lifespan inresponse to calorie restriction (Lin, et al. (2000) Science289:2126-2128; Lin, et al. (2002) Nature 418:344-348). NAD+ is consumedby Sir2 to produce a mixture of 2′- and 3′ O-acetylated ADP-ribose plusnicotinamide and the deacetylated polypeptide (Sauve, et al. (2001)Biochemistry 40:15456-15463). Additional enzymes, includingpoly(ADPribose) polymerases and cADPribose synthases are alsoNAD⁺-dependent and produce nicotinamide and ADPribosyl products (Ziegler(2000) Eur. J. Biochem. 267:1550-1564; Burkle (2001) Bioessays23:795-806).

The non-coenzymatic properties of NAD+ has renewed interest in NAD+biosynthesis. Four recent publications have suggested what is consideredto be all of the gene products and pathways to NAD+ in S. cerevisiae(Panozzo, et al. (2002) FEBS Lett. 517:97-102; Sandmeier, et al. (2002)Genetics 160:877-889; Bitterman, et al. (2002) J. Biol. Chem.277:45099-45107; Anderson, et al. (2003) Nature 423:181-185) depictingconvergence of the flux to NAD+ from de novo synthesis, nicotinic acidimport, and nicotinamide salvage at NaMN (Scheme 1).

SUMMARY OF THE INVENTION

It has now been shown that nicotinamide riboside, which was known to bean NAD+ precursor in bacteria such as Haemophilus influenza (Gingrichand Schlenk (1944) J. Bacteriol. 47:535-550; Leder and Handler (1951) J.Biol. Chem. 189:889-899; Shifrine and Biberstein (1960) Nature 187:623)that lack the enzymes of the de novo and Preiss-Handler pathways(Fleischmann, et al. (1995) Science 269:496-512), is an NAD+ precursorin a previously unknown but conserved eukaryotic NAD+ biosyntheticpathway. Yeast nicotinamide riboside kinase, Nrk1, and human Nrk enzymeswith specific functions in NAD+ metabolism are provided herein. Thespecificity of these enzymes indicates that they are the long-soughttiazofurin kinases that perform the first step in converting cancerdrugs such as tiazofurin and benzamide riboside and their analogs intotoxic NAD+ analogs. Further, yeast mutants of defined genotype were usedto identify sources of nicotinamide riboside and it is shown that milkis a source of nicotinamide riboside.

Accordingly, the present invention is an isolated nucleic acid encodinga eukaryotic nicotinamide riboside kinase polypeptide. A eukaryoticnicotinamide riboside kinase nucleic acid encompasses (a) a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; (b) a nucleotidesequence that hybridizes to a nucleotide sequence of SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:3 or its complementary nucleotide sequence understringent conditions, wherein said nucleotide sequence encodes afunctional nicotinamide riboside kinase polypeptide; or (c) a nucleotidesequence encoding an amino acid sequence encoded by the nucleotidesequences of (a) or (b), but which has a different nucleotide sequencethan the nucleotide sequences of (a) or (b) due to the degeneracy of thegenetic code or the presence of non-translated nucleotide sequences.

The present invention is also an expression vector containing anisolated nucleic acid encoding a eukaryotic nicotinamide riboside kinasepolypeptide. In one embodiment, the expression vector is part of acomposition containing a pharmaceutically acceptable carrier. In anotherembodiment, the composition further contains a prodrug wherein theprodrug is a nicotinamide riboside-related analog that is phosphorylatedby the expressed nicotinamide riboside kinase thereby performing thefirst step in activating said prodrug.

The present invention is also an isolated eukaryotic nicotinamideriboside kinase polypeptide. In one embodiment, the isolatednicotinamide riboside kinase polypeptide has an amino acid sequencehaving at least about 70% amino acid sequence similarity to an aminoacid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or a functionalfragment thereof.

The present invention is further a cultured cell containing an isolatednucleic acid encoding a eukaryotic nicotinamide riboside kinasepolypeptide or a polypeptide encoded thereby.

Still further, the present invention is a composition containing anisolated eukaryotic nicotinamide riboside kinase polypeptide and apharmaceutically acceptable carrier. In one embodiment, the compositionfurther contains a prodrug wherein said prodrug is a nicotinamideriboside-related analog that is phosphorylated by the nicotinamideriboside kinase thereby performing the first step in activating saidprodrug.

The present invention is also a method for treating cancer byadministering to a patient having or suspected of having cancer aneffective amount of a nicotinamide riboside-related prodrug incombination with an isolated eukaryotic nicotinamide riboside kinasepolypeptide or expression vector containing an isolated nucleic acidsequence encoding an eukaryotic nicotinamide riboside kinase polypeptidewherein the nicotinamide riboside kinase polypeptide phosphorylates theprodrug thereby performing the first step in activating the prodrug sothat the signs or symptoms of said cancer are decreased or eliminated.

The present invention is further a method for identifying a natural orsynthetic source for nicotinamide riboside. The method involvescontacting a first cell lacking a functional glutamine-dependent NAD+synthetase with an isolated extract from a natural source or synthetic;contacting a second cell lacking functional glutamine-dependent NAD+synthetase and nicotinamide riboside kinase with the isolated extract;and detecting growth of the first cell compared to the growth of thesecond cell, wherein the presence of growth in the first cell andabsence of growth in the second cell is indicative of the presence ofnicotinamide riboside in the isolated extract. In one embodiment, thenatural source is cow's milk.

Further, the present invention is a dietary supplement compositioncontaining nicotinamide riboside identified in accordance with themethods of the present invention and a carrier.

Moreover, the present invention is a method for preventing or treating adisease or condition associated with the nicotinamide riboside kinasepathway of NAD+ biosynthesis. The method involves administering to apatient having a disease or condition associated with the nicotinamideriboside kinase pathway of NAD+ biosynthesis an effective amount of anicotinamide riboside composition so that the signs or symptoms of thedisease or condition are prevented or reduced. In one embodiment, thenicotinamide riboside is neuroprotective. In another embodiment thenicotinamide riboside is anti-fungal. In a further embodiment, thenicotinamide riboside is administered in combination with tryptophan,nicotinic acid or nicotinamide.

The present invention is also an in vitro method for identifying anicotinamide riboside-related prodrug. The method involves contacting anicotinamide riboside kinase polypeptide with a nicotinamideriboside-related test agent and determining whether said test agent isphosphorylated by said nicotinamide riboside kinase polypeptide whereinphosphorylation of said test agent is indicative of said test agentbeing a nicotinamide riboside-related prodrug. A nicotinamideriboside-related prodrug identified by this method is also encompassedwithin the present invention.

The present invention is further a cell-based method for identifying anicotinamide riboside-related prodrug. This method involves contacting afirst test cell which expresses a recombinant Nrk polypeptide with anicotinamide riboside-related test agent; contacting a second test cellwhich lacks a functional Nrk polypeptide with the same test agent; anddetermining the viability of the first and second test cells, whereinsensitivity of the first cell and not the second cell is indicative of anicotinamide riboside-related prodrug. A nicotinamide riboside-relatedprodrug identified by this method is also encompassed within the contextof the present invention.

The present invention is also a method for identifying an individual ortumor which is susceptible to treatment with a nicotinamideriboside-related prodrug. This method involves detecting the presence ofmutations in, or the level of expression of, a nicotinamide ribosidekinase in an individual or tumor wherein the presence of a mutation orchange in expression of nicotinamide riboside kinase in said individualor tumor compared to a control is indicative of said individual or tumorhaving an altered level of susceptibility to treatment with anicotinamide riboside-related prodrug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence alignment and consensus sequence(SEQ ID NO:34) of human Nrk1 (SEQ ID NO:5), human Nrk2 (SEQ ID NO:6), S.cerevisiae Nrk1 (SEQ ID NO:4), S. pombe nrk1 (SEQ ID NO:7), as comparedto portions of S. cerevisiae uridine/cytidine kinase Urk1 (SEQ ID NO:8)and E. coli pantothenate kinase (SEQ ID NO:9).

DETAILED DESCRIPTION OF THE INVENTION

A Saccharomyces cerevisiae QNS1 gene encoding glutamine-dependent NAD+synthetase has been characterized and mutation of either the glutaminaseactive site or the NAD+ synthetase active site resulted in inviablecells (Bieganowski, et al. (2003) J. Biol. Chem. 278:33049-33055).Possession of strains containing the qns1 deletion and a plasmid-borneQNS1 gene allowed a determination of whether the canonical de novo,import and salvage pathways for NAD+ of Scheme 1 (Panozzo, et al. (2002)supra; Sandmeier, et al. (2002) supra; Bitterman, et al. (2002) supra;Anderson, et al. (2003) supra) are a complete representation of themetabolic pathways to NAD+ in S. cerevisiae. The pathways depicted inscheme 1 suggest that: nicotinamide is deamidated to nicotinic acidbefore the pyridine ring is salvaged to make more NAD+, thussupplementation with nicotinamide may not rescue qns1 mutants byshunting nicotinamide-containing precursors through the pathway; andQNS1 is common to the three pathways, thus there may be no NAD+precursor that rescues qns1 mutants. However, it has now been found thatwhile nicotinamide does not rescue qns1 mutants even at 1 or 10 mM,nicotinamide riboside functions as a vitamin form of NAD+ at 10 μM.

Anticancer agents such as tiazofurin (Cooney, et al. (1983) Adv. EnzymeRegul. 21:271-303) and benzamide riboside (Krohn, et al. (1992) J. Med.Chem. 35:511-517) have been shown to be metabolized intracellularly toNAD+ analogs, taizofurin adenine dinucleotide and benzamide adeninedinucleotide, which inhibit IMP dehydrogenase the rate-limiting enzymefor de novo purine nucleotide biosynthesis.

Though an NMN/NaMN adenylyltransferase is thought to be the enzyme thatconverts the mononucleotide intermediates to NAD+ analogs and thestructural basis for this is known (Zhou et al. (2002) supra), severaldifferent enzymes including adenosine kinase, 5′ nucleotidase (Fridland,et al. (1986) Cancer Res. 46:532-537; Saunders, et al. (1990) CancerRes. 50:5269-5274) and a specific nicotinamide riboside kinase(Saunders, et al. (1990) supra) have been proposed to be responsible fortiazofurin phosphorylation in vivo. A putative nicotinamide ribosidekinase (Nrk) activity was purified, however no amino acid sequenceinformation was obtained and, as a consequence, no genetic test wasperformed to assess its function (Sasiak and Saunders (1996) Arch.Biochem. Biophys. 333:414-418).

Using a qns1 deletion strain that was additionally deleted for yeasthomologs of candidate genes encoding nucleoside kinases proposed tophosphorylate tiazofurin, i.e., adenosine kinase ado1 (Lecoq, et al.(2001) Yeast 18:335-342), uridine/cytidine kinase urk1 (Kern (1990)Nucleic Acids Res. 18:5279; Kurtz, et al. (1999) Curr. Genet.36:130-136), and ribokinase rbk1 (Thierry, et al. (1990) Yeast6:521-534), it was determined whether the nucleoside kinases areuniquely or collectively responsible for utilization of nicotinamideriboside. It was found that despite these deletions, the strain retainedthe ability to utilize nicotinamide riboside in an anabolic pathwayindependent of NAD+ synthetase.

Given that mammalian pharmacology provided no useful clue to theidentity of a putative fungal Nrk, it was considered whether the genemight have been conserved with the Nrk of Haemophilus influenza. The Nrkdomain of H. influenza is encoded by amino acids 225 to 421 of the NadRgene product (the amino terminus of which is NMN adenylyltransferase).Though this domain is structurally similar to yeast thymidylate kinase(Singh, et al. (2002) J. Biol. Chem. 277:33291-33299), sensitivesequence searches revealed that bacterial Nrk has no ortholog in yeast.Genomic searches with the Nrk domain of H. influenza NadR haveidentified a growing list of bacterial genomes predicted to utilizenicotinamide riboside as an NAD+ precursor (Kurnasov, et al. (2002) J.Bacteriol. 184:6906-6917). Thus, had fungi possessed NadR Nrk-homologousdomains, comparative genomics would have already predicted that yeastcan salvage nicotinamide riboside.

To identify the Nrk of S. cerevisiae, an HPLC assay for the enzymaticactivity was established and used in combination with a biochemicalgenomics approach to screen for the gene encoding this activity(Martzen, et al. (1999) Science 286:1153-1155). Sixty-four pools of90-96 S. cerevisiae open reading frames fused to glutathioneS-transferase (GST), expressed in S. cerevisiae, were purified as GSTfusions and screened for the ability to convert nicotinamide ribosideplus ATP to NMN plus ADP. Whereas most pools contained activities thatconsumed some of the input ATP, only pool 37 consumed nicotinamideriboside and produced NMN. In pool 37, approximately half of the 1 mMATP was converted to ADP and the 500 μM nicotinamide riboside peak wasalmost entirely converted to NMN. Examination of the 94 open readingframes that were used to generate pool 37 revealed that YNL129W (SEQ IDNO:1) encodes a predicted 240 amino acid polypeptide with a 187 aminoacid segment containing 23% identity with the 501 amino acid yeasturidine/cytidine kinase Urk1 and remote similarity with a segment of E.coli pantothenate kinase panK (Yun, et al. (2000) J. Biol. Chem.275:28093-28099) (FIG. 1). After cloning YNL129W into a bacterialexpression vector it was ascertained whether this homolog of metabolitekinases was the eukaryotic Nrk. The specific activity of purifiedYNL129W was ˜100-times that of pool 37, consistent with the idea thatall the Nrk activity of pool 37 was encoded by this open reading frame.To test genetically whether this gene product phosphorylatesnicotinamide riboside in vivo, a deletion of YNL129W was created in theqns1 background. It was found that nicotinamide riboside rescue of theqns1 deletion strain was entirely dependent on this gene product. Havingshown biochemically and genetically that YNL129W encodes an authenticNrk activity, the gene was designated NRK1.

A PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402)comparison was conducted on the predicted S. cerevisiae Nrk1 polypeptideand an orthologous human protein Nrk1 (NP_(—)060351; SEQ ID NO:5;FIG. 1) was found. The human NP_(—)060351 protein encoded at locus9q21.31 is a polypeptide of 199 amino acids and is annotated as anuncharacterized protein of the uridine kinase family. In addition, asecond human gene product Nrk2 (NP_(—)733778; SEQ ID NO:6; FIG. 1) wasfound that is 57% identical to human Nrk1. Nrk2 is a 230 amino acidsplice form of what was described as a 186 amino acid muscle integrinbeta 1 binding protein (ITGB1BP3) encoded at 19p13.3 (Li, et al. (1999)J. Cell Biol. 147:1391-1398; Li, et al. (2003) Dev. Biol. 261:209-219).Amino acid conservation between S. cerevisiae, S. pombe and human Nrkhomologs and similarity with fragments of S. cerevisiae Urk1 and E. colipanK is shown in FIG. 1. Fungal and human Nrk enzymes are members of ametabolite kinase superfamily that includes pantothenate kinase but isunrelated to bacterial nicotinamide riboside kinase. Robustcomplementation of the failure of qns1 nrk1 to grow on nicotinamideriboside-supplemented media was provided by human NRK1 and human NRK2cDNA even when expressed from the GAL1 promoter on glucose.

As shown in Table 1, purification of yeast Nrk1 and human Nrk1 and Nrk2revealed high specificity for phosphorylation of nicotinamide ribosideand tiazofurin.

TABLE 1 Nicotinamide riboside Tiazofurin Uridine Cytidine Human Nrk1 275± 17 538 ± 27 19.3 ± 1.7 35.5 ± 6.4 Human Nrk2 2320 ± 20  2150 ± 2102220 ± 170 222 ± 8  Yeast Nrk1 535 ± 60 1129 ± 134 15.2 ± 3.4 82.9 ± 4.4Specific activity is expressed in nmole mg⁻¹ min⁻¹ for phosphorylationof nucleoside substrates.

In the cases of yeast and human Nrk1 enzymes, the enzymes preferredtiazofurin to the natural substrate nicotinamide riboside by a factor oftwo and both enzymes retained less than 7% of their maximal specificactivity on uridine and cytidine. In the case of human Nrk2, the 230amino acid form was essentially equally active on nicotinamide riboside,tiazofurin and uridine with less than 10% of corresponding activity oncytidine. Conversely, the 186 amino acid integrin beta 1 binding proteinform was devoid of enzymatic activity in this in vitro assay and was notfunctional as an Nrk in vivo. However, both the 186 and 230 amino acidisoforms function in vivo in a yeast nicotinamide riboside utilizationassay. Thus, though Nrk2 may contribute additionally to formation ofuridylate, these data demonstrate that fungi and mammals possessspecific nicotinamide riboside kinases that function to synthesize NAD+through NMN in addition to the well-known pathways through NaMN.Identification of Nrk enzymatic activities thus accounts for the dualspecificity of fungal and mammalian NaMN/NMN adenylyltransferases.

On the basis of SAGE data, NRK1 is a rare message in many tissuesexamined while NRK2 is highly expressed in heart and skeletal muscle andhas lower level expression in retinal epithelium and placenta (Boon, etal. (2002) Proc. Natl. Acad. Sci. USA 99:11287-11292). From cancer cellline to cancer cell line the expression levels are quite variable (Boon,et al. (2002) supra). Thus, in individuals whose tumors are NRK1,NRK2-low, tiazofurin conversion to NAD+ may occur more extensively inthe patients hearts and muscles than in tumors. In tumors that are NRK1and/or NRK2-high, a substantial amount of tiazofurin may be converted totiazofurin adenine dinucleotide in tumors.

A yeast qns1 mutant was used to screen for natural sources ofnicotinamide riboside wherein it was identified in an acid wheypreparation of cow's milk. Unlike the original screen for vitamins inprotein-depleted extracts of liver for reversal of black-tongue instarving dogs (Elvehjem, et al. (1938) J. Biol. Chem. 123:137-149), thisassay is pathway-specific in identifying NAD+ precursors. Because of theqns1 deletion, nicotinic acid and nicotinamide do not score positivelyin this assay. As the factor from milk requires nicotinamide ribosidekinase for growth, the nutrient is clearly nicotinamide riboside and notNMN or NAD+.

A revised metabolic scheme for NAD+, incorporating Nrk1 homologs and thenicotinamide riboside salvage pathway is shown in Scheme 2 whereindouble arrows depict metabolic steps common to yeast and humans (withyeast gene names) and single arrows depict steps unique to humans (PBEF,nicotinamide phosphoribosyltransferase) and yeast (Pnc1,nicotinamidase).

A difference between humans and yeasts concerns the organisms' uses ofnicotinamide and nicotinic acid, the two niacins that were co-identifiedas anti-black tongue factor (Elvehjem, et al. (1938) supra). Humansencode a homolog of the Haemophilus ducreyi nadV gene, termed pre-B-cellcolony enhancing factor, that may convert nicotinamide to NMN (Rongvaux,et al. (2002) Eur. J. Immunol. 32:3225-3234) and is highly inducedduring lymphocyte activation (Samal, et al. (1994) Mol. Cell. Biol.14:1431-1437). In contrast, S. cerevisiae lacks a homolog of nadV andinstead has a homolog of the E. coli pncA gene, termed PNC1, thatconverts nicotinamide to nicotinic acid for entry into thePreiss-Handler pathway (Ghislain, et al. (2002) Yeast 19:215-224;Sandmeier, et al. (2002) supra). Though the Preiss-Handler pathway isfrequently considered a salvage pathway from nicotinamide, ittechnically refers to the steps from nicotinic acid to NAD+ (Preiss andHandler (1958) supra; Preiss and Handler (1958) supra). Reports thatnicotinamidase had been purified from mammalian liver in the 1960s(Petrack, et al. (1965) J. Biol. Chem. 240:1725-1730) may havecontributed to the sense that fungal and animal NAD+ biosynthesis isentirely conserved. However, animal genes for nicotinamidase have notbeen identified and there is no compelling evidence that nicotinamideand nicotinic acid are utilized as NAD+ precursors through the sameroute in mammals. The persistence of “niacin” as a mixture ofnicotinamide and nicotinic acid may attest to the utility of utilizingmultiple pathways to generate NAD+ and indicates that supplementationwith nicotinamide riboside as third importable NAD+ precursor can bebeneficial for certain conditions.

First reported in 1955, high doses of nicotinic acid are effective atreducing cholesterol levels (Altschul, et al. (1955) Arch. Biochem.Biophys. 54:558-559). Since the initial report, many controlled clinicalstudies have shown that nicotinic acid preparations, alone and incombination with HMG CoA reductase inhibitors, are effective incontrolling low-density lipoprotein cholesterol, increasing high-densitylipoprotein cholesterol, and reducing triglyceride and lipoprotein alevels in humans (Pasternak, et al. (1996) Ann. Intern. Med.125:529-540). Though nicotinic acid treatment effects all of the keylipids in the desirable direction and has been shown to reduce mortalityin target populations (Pasternak, et al. (1996) supra), its use islimited because of a side effect of heat and redness termed “flushing,”which is significantly effected by the nature of formulation (Capuzzi,et al. (2000) Curr. Atheroscler. Rep. 2:64-71). Thus, nicotinamideriboside supplementation could be one route to improve lipid profiles inhumans. Further, nicotinamide is protective in animal models of stroke(Klaidman, et al. (2003) Pharmacology 69:150-157) and nicotinamideriboside could be an important supplement for acute conditions such asstroke. Additionally, regulation of NAD+ biosynthetic enzymes could beuseful in sensitizing tumors to compounds such as tiazofurin, to protectnormal tissues from the toxicity of compounds such as tiazofurin adeninedinucleotide, and to stratify patients for the most judicious use oftiazofurin chemotherapy.

The present invention is an isolated nucleic acid containing aeukaryotic nucleotide sequence encoding a nicotinamide riboside kinasepolypeptide. As used herein, an isolated molecule (e.g., an isolatednucleic acid such as genomic DNA, RNA or cDNA or an isolatedpolypeptide) means a molecule separated or substantially free from atleast some of the other components of the naturally occurring organism,such as for example, the cell structural components or otherpolypeptides or nucleic acids commonly found associated with themolecule. When the isolated molecule is a polypeptide, said polypeptideis at least about 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% or more pure (w/w).

In one embodiment, the eukaryotic nucleotide sequence encoding anicotinamide riboside kinase polypeptide is a nucleotide sequence of SEQID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In another embodiment, theeukaryotic nucleotide sequence encoding a nicotinamide riboside kinasepolypeptide is a nucleotide sequence that hybridizes to a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or its complementarynucleotide sequence under stringent conditions, wherein said nucleotidesequence encodes a functional nicotinamide riboside kinase polypeptide.In a further embodiment, the eukaryotic nucleotide sequence encoding anicotinamide riboside kinase polypeptide is a nucleotide sequenceencoding a functional nicotinamide riboside kinase polypeptide but whichhas a different nucleotide sequence than the nucleotide sequences of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3 due to the degeneracy of thegenetic code or the presence of non-translated nucleotide sequences.

As used herein, a functional polypeptide is one that retains at leastone biological activity normally associated with that polypeptide.Alternatively, a functional polypeptide retains all of the activitiespossessed by the unmodified peptide. By retains biological activity, itis meant that the polypeptide retains at least about 50%, 60%, 75%, 85%,90%, 95%, 97%, 98%, 99%, or more, of the biological activity of thenative polypeptide (and can even have a higher level of activity thanthe native polypeptide). A non-functional polypeptide is one thatexhibits essentially no detectable biological activity normallyassociated with the polypeptide (e.g., at most, only an insignificantamount, e.g., less than about 10% or even 5%).

As used herein, the term polypeptide encompasses both peptides andproteins, unless indicated otherwise.

A nicotinamide riboside kinase polypeptide or Nrk protein as usedherein, is intended to be construed broadly and encompasses an enzymecapable of phosphorylating nicotinamide riboside. The term nicotinamideriboside kinase or Nrk also includes modified (e.g., mutated) Nrk thatretains biological function (i.e., have at least one biological activityof the native Nrk protein, e.g., phosphorylating nicotinamide riboside),functional Nrk fragments including truncated molecules, alternativelyspliced isoforms (e.g., the alternatively spliced isoforms of humanNrk2), and functional Nrk fusion polypeptides (e.g., an Nrk-GST proteinfusion or Nrk-His tagged protein).

Any Nrk polypeptide or Nrk-encoding nucleic acid known in the art can beused according to the present invention. The Nrk polypeptide orNrk-encoding nucleic acid can be derived from yeast, fungal (e.g.,Saccharomyces cerevisiae, Saccharomyces pombe, Pichia sp., Neurosporasp., and the like) plant, animal (e.g., insect, avian (e.g., chicken),or mammalian (e.g., rat, mouse, bovine, porcine, ovine, caprine, equine,feline, canine, lagomorph, simian, human and the like) sources.

Representative cDNA and amino acid sequences of a S. cerevisiae Nrk1 areshown in SEQ ID NO:1 and SEQ ID NO:4 (FIG. 1), respectively.Representative cDNA and amino acid sequences of a human Nrk1 are shownin SEQ ID NO:2 and SEQ ID NO:5 (FIG. 1), respectively. RepresentativecDNA and amino acid sequences of a human Nrk2 are shown in SEQ ID NO:3and SEQ ID NO:6 (FIG. 1), respectively. Other Nrk sequences encompassedby the present invention include, but are not limited to, Nrk1 ofGENBANK accession numbers NM_(—)017881, AK000566, BC001366, BC036804,and BC026243 and Nrk2 of GENBANK accession number NM_(—)170678.Moreover, locus CAG61927 from the Candida glabrata CBS138 genome project(Dujon, et al. (2004) Nature 430:35-44) is 54% identical to theSaccharomyces cerevisiae Nrk1 protein. Particular embodiments of thepresent invention embrace a Nrk polypeptide having the conserved aminoacid sequence XXXXDDFXK (SEQ ID NO:34), wherein Xaa₁ and Xaa₂ arealiphatic amino acid residues, Xaa₃ is His or Ser, Xaa₄ is a hydrophilicamino acid residue, and Xaa₅ is an aromatic amino acid residue.

To illustrate, hybridization of such sequences can be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% Formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/or conditionsrepresented by a wash stringency of 50% Formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the sequencesspecifically disclosed herein. See, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring HarborLaboratory).

Alternatively stated, isolated nucleic acids encoding Nrk of theinvention have at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% orhigher sequence similarity with the isolated nucleic acid sequencesspecifically disclosed herein (or fragments thereof, as defined above)and encode a functional Nrk as defined herein.

It will be appreciated by those skilled in the art that there can bevariability in the nucleic acids that encode the Nrk of the presentinvention due to the degeneracy of the genetic code. The degeneracy ofthe genetic code, which allows different nucleic acid sequences to codefor the same polypeptide, is well known in the literature (see Table 2).

TABLE 2 3- 1- Letter Letter Amino Acid Code Code Codons Alanine Ala AGCA GCC GCG GCT Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GATGlutamic acid Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly GGGA GGC GGG GGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC ATTLysine Lys K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT MethionineMet M ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCTGlutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT SerineSer S AGC ACT TCA TCC TCG TCT Threonine Thr T ACA ACC ACG ACT Valine ValV GTA GTC GTG GTT Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

Further variation in the nucleic acid sequence can be introduced by thepresence (or absence) of non-translated sequences, such as intronicsequences and 5′ and 3′ untranslated sequences.

Moreover, the isolated nucleic acids of the invention encompass thosenucleic acids encoding Nrk polypeptides that have at least about 60%,70%, 80%, 90%, 95%, 97%, 98% or higher amino acid sequence similaritywith the polypeptide sequences specifically disclosed herein (orfragments thereof) and further encode a functional Nrk as definedherein.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity orsimilarity to a known sequence. Sequence identity and/or similarity canbe determined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman (1981) Adv. Appl. Math. 2:482, by the sequence identityalignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson & Lipman (1988) Proc.Natl. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux, et al.(1984) Nucl. Acid Res. 12:387-395, either using the default settings, orby inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle (1987) J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins &Sharp (1989) CABIOS 5:151-153.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul, et al. (1990) J. Mol. Biol. 215:403-410 and Karlin, et al.(1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly usefulBLAST program is the WU-BLAST-2 program which was obtained fromAltschul, et al. (1996) Methods in Enzymology, 266:460-480;http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, which can be set to the default values. The parameters aredynamic values and are established by the program itself depending uponthe composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values can be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul,et al. (1997) Nucleic Acids Res. 25:3389-3402.

A percentage amino acid sequence identity value can be determined by thenumber of matching identical residues divided by the total number ofresidues of the longer sequence in the aligned region. The longersequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the shortersequence in the aligned region and multiplying by 100. The longersequence is the one having the most actual residues in the alignedregion.

To modify Nrk amino acid sequences specifically disclosed herein orotherwise known in the art, amino acid substitutions can be based on anycharacteristic known in the art, including the relative similarity ordifferences of the amino acid side-chain substituents, for example,their hydrophobicity, hydrophilicity, charge, size, and the like. Inparticular embodiments, conservative substitutions (i.e., substitutionwith an amino acid residue having similar properties) are made in theamino acid sequence encoding Nrk.

In making amino acid substitutions, the hydropathic index of amino acidsmay be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (see, Kyte and Doolittle (1982) J. Mol. Biol.157:105). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle (1982)supra), and these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Isolated nucleic acids of this invention include RNA, DNA (includingcDNAs) and chimeras thereof. The isolated nucleic acids can furthercontain modified nucleotides or nucleotide analogs.

The isolated nucleic acids encoding Nrk can be associated withappropriate expression control sequences, e.g.,transcription/translation control signals and polyadenylation signals.

It will be appreciated that a variety of promoter/enhancer elements canbe used depending on the level and tissue-specific expression desired.The promoter can be constitutive or inducible (e.g., the metallothioneinpromoter or a hormone inducible promoter), depending on the pattern ofexpression desired. The promoter can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Thepromoter is chosen so that it will function in the target cell(s) ofinterest. In particular embodiments, the promoter functions in tumorcells or in cells that can be used to express nucleic acids encoding Nrkfor the purposes of large-scale protein production. Likewise, thepromoter can be specific for these cells and tissues (i.e., only showsignificant activity in the specific cell or tissue type).

To illustrate, an Nrk coding sequence can be operatively associated witha cytomegalovirus (CMV) major immediate-early promoter, an albuminpromoter, an Elongation Factor 1-α (EF1-α) promoter, a PγK promoter, aMFG promoter, a Rous sarcoma virus promoter, or aglyceraldehyde-3-phosphate promoter.

Moreover, specific initiation signals are generally required forefficient translation of inserted protein coding sequences. Thesetranslational control sequences, which can include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

Nrk can be expressed not only directly, but also as a fusion proteinwith a heterologous polypeptide, i.e. a signal sequence for secretionand/or other polypeptide which will aid in the purification of Nrk. Inone embodiment, the heterologous polypeptide has a specific cleavagesite to remove the heterologous polypeptide from Nrk.

In general, a signal sequence can be a component of the vector andshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For production in a prokaryote, aprokaryotic signal sequence from, for example, alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders can be used.For yeast secretion, one can use, e.g., the yeast invertase, alphafactor, or acid phosphatase leaders, the Candida albicans glucoamylaseleader (EP 362,179), or the like (see, for example WO 90/13646). Inmammalian cell expression, signal sequences from secreted polypeptidesof the same or related species, as well as viral secretory leaders, forexample, the herpes simplex glycoprotein D signal can be used.

Other useful heterologous polypeptides which can be fused to Nrk includethose which increase expression or solubility of the fusion protein oraid in the purification of the fusion protein by acting as a ligand inaffinity purification. Typical fusion expression vectors include thoseexemplified herein as well as pMAL (New England Biolabs, Beverly, Mass.)and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse maltose E bindingprotein or protein A, respectively, to the target recombinant protein.

The isolated nucleic acids encoding Nrk can be incorporated into avector, e.g., for the purposes of cloning or other laboratorymanipulations, recombinant protein production, or gene delivery. Inparticular embodiments, the vector is an expression vector. Exemplaryvectors include bacterial artificial chromosomes, cosmids, yeastartificial chromosomes, phage, plasmids, lipid vectors and viralvectors. By the term express, expresses or expression of a nucleic acidcoding sequence, in particular an Nrk coding sequence, it is meant thatthe sequence is transcribed, and optionally, translated. Typically,according to the present invention, transcription and translation of thecoding sequence will result in production of Nrk polypeptide.

The methods of the present invention provide a means for delivering, andoptionally expressing, nucleic acids encoding Nrk in a broad range ofhost cells, including both dividing and non-dividing cells in vitro(e.g., for large-scale recombinant protein production or for use inscreening assays) or in vivo (e.g., for recombinant large-scale proteinproduction, for creating an animal model for disease, or for therapeuticpurposes). In embodiments of the invention, the nucleic acid can beexpressed transiently in the target cell or the nucleic acid can bestably incorporated into the target cell, for example, by integrationinto the genome of the cell or by persistent expression from stablymaintained episomes (e.g., derived from Epstein Barr Virus).

The isolated nucleic acids, vectors, methods and pharmaceuticalformulations of the present invention find use in a method ofadministering a nucleic acid encoding Nrk to a subject. In this manner,Nrk can thus be produced in vivo in the subject. The subject can have adeficiency of Nrk, or the production of a foreign Nrk in the subject canimpart some therapeutic effect. Pharmaceutical formulations and methodsof delivering nucleic acids encoding Nrk for therapeutic purposes aredescribed herein.

Alternatively, an isolated nucleic acid encoding Nrk can be administeredto a subject so that the nucleic acid is expressed by the subject andNrk is produced and purified therefrom, i.e., as a source of recombinantNrk protein. According to this embodiment, the Nrk is secreted into thesystemic circulation or into another body fluid (e.g., milk, lymph,spinal fluid, urine) that is easily collected and from which the Nrk canbe further purified. As a further alternative, Nrk protein can beproduced in avian species and deposited in, and conveniently isolatedfrom, egg proteins.

Likewise, Nrk-encoding nucleic acids can be expressed transiently orstably in a cell culture system for the purpose of screening assays orfor large-scale recombinant protein production. The cell can be abacterial, protozoan, plant, yeast, fungus, or animal cell. In oneembodiment, the cell is an animal cell (e.g., insect, avian ormammalian), and in another embodiment a mammalian cell (e.g., afibroblast).

It will be apparent to those skilled in the art that any suitable vectorcan be used to deliver the isolated nucleic acids of this invention tothe target cell(s) or subject of interest. The choice of delivery vectorcan be made based on a number of factors known in the art, including ageand species of the target host, in vitro vs. in vivo delivery, level andpersistence of expression desired, intended purpose (e.g., for therapyor drug screening), the target cell or organ, route of delivery, size ofthe isolated nucleic acid, safety concerns, and the like.

Suitable vectors include virus vectors (e.g., retrovirus, alphavirus;vaccinia virus; adenovirus, adeno-associated virus, or herpes simplexvirus), lipid vectors, poly-lysine vectors, synthetic polyamino polymervectors that are used with nucleic acid molecules, such as plasmids, andthe like.

As used herein, the term viral vector or viral delivery vector can referto a virus particle that functions as a nucleic acid delivery vehicle,and which contains the vector genome packaged within a virion.Alternatively, these terms can be used to refer to the vector genomewhen used as a nucleic acid delivery vehicle in the absence of thevirion.

Protocols for producing recombinant viral vectors and for using viralvectors for nucleic acid delivery can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989) and other standard laboratory manuals (e.g., Vectorsfor Gene Therapy. In: Current Protocols in Human Genetics. John Wileyand Sons, Inc.: 1997).

Particular examples of viral vectors are those previously employed forthe delivery of nucleic acids including, for example, retrovirus,adenovirus, AAV, herpes virus, and poxvirus vectors.

In certain embodiments of the present invention, the delivery vector isan adenovirus vector. The term adenovirus as used herein is intended toencompass all adenoviruses, including the Mastadenovirus andAviadenovirus genera. To date, at least forty-seven human serotypes ofadenoviruses have been identified (see, e.g., Fields, et al., Virology,volume 2, chapter 67 (3d ed., Lippincott-Raven Publishers). In oneembodiment, the adenovirus is a human serogroup C adenovirus, in anotherembodiment the adenovirus is serotype 2 (Ad2) or serotype 5 (Ad5) orsimian adenovirus such as AdC68.

Those skilled in the art will appreciate that vectors can be modified ortargeted as described in Douglas, et al. (1996) Nature Biotechnology14:1574 and U.S. Pat. Nos. 5,922,315; 5,770,442 and/or 5,712,136.

An adenovirus genome can be manipulated such that it encodes andexpresses a nucleic acid of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner, et al. (1988) BioTechniques 6:616; Rosenfeld, et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.

Recombinant adenoviruses can be advantageous in certain circumstances inthat they are not capable of infecting nondividing cells and can be usedto infect a wide variety of cell types, including epithelial cells.Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., as occurswith retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large relative to other delivery vectors(Haj-Ahmand and Graham (1986) J. Virol. 57:267).

In particular embodiments, the adenovirus genome contains a deletiontherein, so that at least one of the adenovirus genomic regions does notencode a functional protein. For example, an adenovirus vectors can haveE1 genes and packaged using a cell that expresses the E1 proteins (e.g.,293 cells). The E3 region is also frequently deleted as well, as thereis no need for complementation of this deletion. In addition, deletionsin the E4, E2a, protein IX, and fiber protein regions have beendescribed, e.g., by Armentano, et al. (1997) J. Virology 71:2408; Gao,et al. (1996) J. Virology 70:8934; Dedieu, et al. (1997) J. Virology71:4626; Wang, et al. (1997) Gene Therapy 4:393; U.S. Pat. No.5,882,877. In general, the deletions are selected to avoid toxicity tothe packaging cell. Combinations of deletions that avoid toxicity orother deleterious effects on the host cell can be routinely selected bythose skilled in the art.

Those skilled in the art will appreciate that typically, with theexception of the E3 genes, any deletions will need to be complemented inorder to propagate (replicate and package) additional virus, e.g., bytranscomplementation with a packaging cell.

The present invention can also be practiced with gutted adenovirusvectors (as that term is understood in the art, see e.g., Lieber, et al.(1996) J. Virol. 70:8944-60) in which essentially all of the adenovirusgenomic sequences are deleted.

Adeno-associated viruses (AAV) have also been employed as nucleic aciddelivery vectors. For a review, see Muzyczka et al. Curr. Topics inMicro. and Immunol. (1992) 158:97-129). AAV are among the few virusesthat can integrate their DNA into non-dividing cells, and exhibit a highfrequency of stable integration into human chromosome (see, for example,Flotte, et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski, et al., (1989) J. Virol. 63:3822-3828; McLaughlin, et al.(1989) J. Virol. 62:1963-1973). A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see, forexample, Hermonat, et al. (1984) Proc. Natl. Acad. Sci. USA81:6466-6470; Tratschin, et al. (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford, et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin, et al.(1984) J. Virol. 51:611-619; and Flotte, et al. (1993) J. Biol. Chem.268:3781-3790).

Any suitable method known in the art can be used to produce AAV vectorsexpressing the nucleic acids encoding Nrk of this invention (see, e.g.,U.S. Pat. Nos. 5,139,941; 5,858,775; 6,146,874 for illustrativemethods). In one particular method, AAV stocks can be produced byco-transfection of a rep/cap vector encoding AAV packaging functions andthe template encoding the AAV vDNA into human cells infected with thehelper adenovirus (Samulski, et al. (1989) J. Virology 63:3822). The AAVrep and/or cap genes can alternatively be provided by a packaging cellthat stably expresses the genes (see, e.g., Gao, et al. (1998) HumanGene Therapy 9:2353; Inoue, et al. (1998) J. Virol. 72:7024; U.S. Pat.No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947).

Another vector for use in the present invention is Herpes Simplex Virus(HSV). HSV can be modified for the delivery of nucleic acids to cells byproducing a vector that exhibits only the latent function for long-termgene maintenance. HSV vectors are useful for nucleic acid deliverybecause they allow for a large DNA insert of up to or greater than 20kilobases; they can be produced with extremely high titers; and theyhave been shown to express nucleic acids for a long period of time inthe central nervous system as long as the lytic cycle does not occur.

In other particular embodiments of the present invention, the deliveryvector of interest is a retrovirus. The development of specialized celllines (termed packaging cells) which produce only replication-defectiveretroviruses has increased the utility of retroviruses for gene therapy,and defective retroviruses are characterized for use in gene transferfor gene therapy purposes (for a review, see Miller (1990) Blood76:271). A replication-defective retrovirus can be packaged into virionswhich can be used to infect a target cell through the use of a helpervirus by standard techniques.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed. Many non-viral methods ofnucleic acid transfer rely on normal mechanisms used by mammalian cellsfor the uptake and intracellular transport of macromolecules. Inparticular embodiments, non-viral nucleic acid delivery systems rely onendocytic pathways for the uptake of the nucleic acid molecule by thetargeted cell. Exemplary nucleic acid delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In particular embodiments, plasmid vectors are used in the practice ofthe present invention. Naked plasmids can be introduced into musclecells by injection into the tissue. Expression can extend over manymonths, although the number of positive cells is typically low (Wolff,et al. (1989) Science 247:247). Cationic lipids have been demonstratedto aid in introduction of nucleic acids into some cells in culture(Feigner and Ringold (1989) Nature 337:387). Injection of cationic lipidplasmid DNA complexes into the circulation of mice has been shown toresult in expression of the DNA in lung (Brigham, et al. (1989) Am. J.Med. Sci. 298:278). One advantage of plasmid DNA is that it can beintroduced into non-replicating cells.

In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) can be entrapped in a lipid particle bearing positive chargeson its surface and, optionally, tagged with antibodies againstcell-surface antigens of the target tissue (Mizuno, et al. (1992) NoShinkei Geka 20:547; WO 91/06309; Japanese patent application 1047381;and European patent publication EP-A-43075).

Liposomes that consist of amphiphilic cationic molecules are usefulnon-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal (1995) Science 270:404-410; Blaese, et al. (1995)Cancer Gene Ther. 2:291-297; Behr, et al. (1994) Bioconjugate Chem.5:382-389; Remy, et al. (1994) Bioconjugate Chem. 5:647-654; and Gao, etal. (1995) Gene Therapy 2:710-722). The positively charged liposomes arebelieved to complex with negatively charged nucleic acids viaelectrostatic interactions to form lipid:nucleic acid complexes. Thelipid:nucleic acid complexes have several advantages as nucleic acidtransfer vectors. Unlike viral vectors, the lipid:nucleic acid complexescan be used to transfer expression cassettes of essentially unlimitedsize. Since the complexes lack proteins, they can evoke fewerimmunogenic and inflammatory responses. Moreover, they cannot replicateor recombine to form an infectious agent and have low integrationfrequency. A number of publications have demonstrated that amphiphiliccationic lipids can mediate nucleic acid delivery in vivo and in vitro(Felgner, et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-17; Loeffler,et al. (1993) Methods in Enzymology 217:599-618; Feigner, et al. (1994)J. Biol. Chem. 269:2550-2561).

As indicated above, Nrk polypeptide can be produced in, and optionallypurified from, cultured cells or organisms expressing a nucleic acidencoding Nrk for a variety of purposes (e.g., screening assays,large-scale protein production, therapeutic methods based on delivery ofpurified Nrk).

In particular embodiments, an isolated nucleic acid encoding Nrk can beintroduced into a cultured cell, e.g., a cell of a primary orimmortalized cell line for recombinant protein production. Therecombinant cells can be used to produce the Nrk polypeptide, which iscollected from the cells or cell culture medium. Likewise, recombinantprotein can be produced in, and optionally purified from an organism(e.g., a microorganism, animal or plant) being used essentially as abioreactor.

Generally, the isolated nucleic acid is incorporated into an expressionvector (viral or nonviral as described herein). Expression vectorscompatible with various host cells are well-known in the art and containsuitable elements for transcription and translation of nucleic acids.Typically, an expression vector contains an expression cassette, whichincludes, in the 5′ to 3′ direction, a promoter, a coding sequenceencoding an Nrk operatively associated with the promoter, and,optionally, a termination sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system), yeast cells or mammalian cells. Somesuitable host cells are discussed further in Goeddel (1990) GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al. (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz, et al.(1987) Gene 54:113-123), and pYES2 (INVITROGEN Corporation, San Diego,Calif.). Baculovirus vectors available for expression of nucleic acidsto produce proteins in cultured insect cells (e.g., Sf 9 cells) includethe pAc series (Smith, et al. (1983) Mol. Cell. Biol. 3:2156-2165) andthe pVL series (Lucklow and Summers (1989) Virology 170:31-39).

Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329:840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand Simian Virus 40.

In addition to the regulatory control sequences discussed herein, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector can encode aselectable marker gene to identify host cells that have incorporated thevector.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms transformation and transfection refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNAcomplexes, cell sonication, gene bombardment using high velocitymicroprojectiles, and viral-mediated transfection. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

Often only a small fraction of cells (in particular, mammalian cells)integrate the foreign DNA into their genome. In order to identify andselect these integrants, a nucleic acid that encodes a selectable marker(e.g., resistance to antibiotics) can be introduced into the host cellsalong with the nucleic acid of interest. In particular embodiments,selectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acids encoding aselectable marker can be introduced into a host cell on the same vectoras that comprising the nucleic acid of interest or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

Recombinant proteins can also be produced in a transgenic plant in whichthe isolated nucleic acid encoding the protein is inserted into thenuclear or plastidic genome. Plant transformation is known as the art.See, in general, Methods in Enzymology Vol. 153 (Recombinant DNA Part D)1987, Wu and Grossman Eds., Academic Press and European PatentApplication EP 693554.

The present invention further provides cultured or recombinant cellscontaining the isolated nucleic acids encoding Nrk for use in thescreening methods and large-scale protein production methods of theinvention (e.g., Nrk is produced and collected from the cells and,optionally, purified). In one particular embodiment, the inventionprovides a cultured cell containing an isolated nucleic acid encodingNrk as described above for use in a screening assay for identifying anicotinamide riboside-related prodrug. Also provided is a cell in vivoproduced by a method comprising administering an isolated nucleic acidencoding Nrk to a subject in a therapeutically effective amount.

For in vitro screening assays and therapeutic administration, Nrkpolypeptides can be purified from cultured cells. Typically, thepolypeptide is recovered from the culture medium as a secretedpolypeptide, although it also can be recovered from host cell lysateswhen directly expressed without a secretory signal. When Nrk isexpressed in a recombinant cell other than one of human origin, the Nrkis completely free of proteins or polypeptides of human origin. However,it is necessary to purify Nrk from recombinant cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to Nrk. As a first step, the culture medium or lysate is centrifugedto remove particulate cell debris. The membrane and soluble proteinfractions are then separated. The Nrk can then be purified from thesoluble protein fraction. Nrk thereafter can then be purified fromcontaminant soluble proteins and polypeptides with, for example, thefollowing suitable purification procedures: by fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, SEPHADEX G-75; ligand affinitychromatography, and protein A SEPHAROSE columns to remove contaminantssuch as IgG.

As Nrk phosphorylates tiazofurin, thereby performing the first step inactivating it, Nrk is a useful target for identifying compounds whichupon phosphorylation by Nrk and subsequent adenylylation inhibit IMPDH.As it has been shown that inhibitors of the IMPDH enzyme function asanti-bovine viral diarrhoea virus agents (Stuyver, et al. (2002)Antivir. Chem. Chemother. 13(6):345-52); inhibitors of IMPDH blockhepatitis B replicon colony-forming efficiency (Zhou, et al. (2003)Virology 310(2):333-42); and tiazofurin (Cooney, et al. (1983) Adv.Enzyme Regul. 21:271-303) and benzamide riboside (Krohn, et al. (1992)J. Med. Chem. 35:511-517), when activated, inhibit IMP dehydrogenase; itis contemplated by using Nrk and the nicotinamide riboside pathway fordrug screening, anticancer and antiviral agents will be identified.Accordingly, the present invention provides methods for identifying anicotinamide riboside-related prodrug. As used herein, a nicotinamideriboside-related prodrug is any analog of nicotinamide riboside (e.g.,tiazofurin and benzamide riboside) that, when phosphorylated by Nrk,ultimately can result in cell death or antiviral activity.

In one embodiment, a nicotinamide riboside-related prodrug is identifiedin a cell-free assay using isolated Nrk polypeptide. The steps involvedin a this screening assay of the invention include, isolating orpurifying an Nrk polypeptide; contacting or adding at least onenicotinamide riboside-related test agent to a point of application, suchas a well, in the plate containing the isolated Nrk and a suitablephosphate donor such as ATP, Mg-ATP, Mn-ATP, Mg-GTP or Mn-GTP; anddetermining whether said test agent is phosphorylated by said Nrkpolypeptide wherein phosphorylation of said test agent is indicative ofa nicotinamide riboside-related prodrug. The phosphate donor can beadded with or after the agent and the assay can be carried out undersuitable assay conditions for phosphorylation, such as those exemplifiedherein.

With respect to the cell-free assay, test agents can be synthesized orotherwise affixed to a solid substrate, such as plastic pins, glassslides, plastic wells, and the like. Further, isolated Nrk can be freein solution, affixed to a solid support, or expressed on a cell surface.

Alternatively, an Nrk fusion protein can be provided to facilitatebinding of Nrk to a matrix. For example, glutathione-S-transferasefusion proteins can be adsorbed onto glutathione SEPHAROSE beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the test agent, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH) and phosphorylation as described above.

In another embodiment, a nicotinamide riboside-related prodrug isidentified in a cell-based assay. The steps involved in a this screeningassay of the invention include, contacting a first test cell whichexpresses a recombinant Nrk polypeptide with a nicotinamideriboside-related test agent; contacting a second test cell which lacks afunctional Nrk polypeptide with the same test agent; and determining theviability of the first and second test cells wherein sensitivity or celldeath of the first cell and not the second cell is indicative of anicotinamide riboside-related prodrug. While the cell-based assay can becarried out using any suitable cell including bacteria, yeast, insectcells (e.g., with a baculovirus expression system), avian cells,mammalian cells, or plant cells, in particular embodiments, the testcell is a mammalian cell. In a further embodiment, said cell lacks afunctional endogenous Nrk (e.g., the endogenous Nrk has been deleted ormutated or the cell does not express an Nrk). Said first test cell istransformed or transfected with an expression vector containing anexogenous Nrk so that upon exposure to a test agent, viability of thetransformed cell can be compared to a second test cell lacking any Nrkactivity. Thus, it can be ascertained whether the test agent is beingactivated in an Nrk-dependent manner. Cells modified to express arecombinant Nrk can be transiently or stably transformed with thenucleic acid encoding Nrk. Stably transformed cells can be generated bystable integration into the genome of the organism or by expression froma stably maintained episome (e.g., Epstein Barr Virus derived episomes).

Suitable methods for determining cell viability are well-established inthe art. One such method uses non-permeant dyes (e.g., propidium iodide,7-Amino Actinomycin D) that do not enter cells with intact cellmembranes or active cell metabolism. Cells with damaged plasma membranesor with impaired/no cell metabolism are unable to prevent the dye fromentering the cell. Once inside the cell, the dyes bind to intracellularstructures producing highly fluorescent adducts which identify the cellsas non-viable. Alternatively, cell viability can be determined byassaying for active cell metabolism which results in the conversion of anon-fluorescent substrate into a highly fluorescent product (e.g.,fluorescein diacetate).

The test cells of the screening method of the invention can be culturedunder standard conditions of temperature, incubation time, opticaldensity, plating density and media composition corresponding to thenutritional and physiological requirements of the cells. However,conditions for maintenance and growth of the test cell can be differentfrom those for assaying candidate agents in the screening methods of theinvention. Any techniques known in the art can be applied to establishthe optimal conditions.

Screening assays of the invention can be performed in any format thatallows rapid preparation and processing of multiple reactions such asin, for example, multi-well plates of the 96-well variety. Stocksolutions of the agents as well as assay components are preparedmanually and all subsequent pipetting, diluting, mixing, washing,incubating, sample readout and data collecting is done usingcommercially available robotic pipetting equipment, automated workstations, and analytical instruments for detecting the output of theassay.

In addition to the reagents provided above, a variety of other reagentscan be included in the screening assays of the invention. These includereagents like salts, neutral proteins, e.g., albumin, detergents, etc.Also, reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents, andthe like can be used.

Screening assays can also be carried out in vivo in animals. Thus, thepresent invention provides a transgenic non-human animal containing anisolated nucleic acid encoding Nrk, which can be produced according tomethods well-known in the art. The transgenic non-human animal can beany species, including avians and non-human mammals. IN accordance withthe invention, suitable non-human mammals include mice, rats, rabbits,guinea pigs, goats, sheep, pigs and cattle. Mammalian models for cancer,bovine diarrhoea viral infection or hepatitis C viral infection can alsobe used.

A nucleic acid encoding Nrk is stably incorporated into cells within thetransgenic animal (typically, by stable integration into the genome orby stably maintained episomal constructs). It is not necessary thatevery cell contain the transgene, and the animal can be a chimera ofmodified and unmodified cells, as long as a sufficient number of cellscontain and express the Nrk transgene so that the animal is a usefulscreening tool (e.g., so that administration of test agents give rise todetectable cell death or anti-viral activity).

Methods of making transgenic animals are known in the art. DNAconstructs can be introduced into the germ line of an avian or mammal tomake a transgenic animal. For example, one or several copies of theconstruct can be incorporated into the genome of an embryo by standardtransgenic techniques.

In an exemplary embodiment, a transgenic non-human animal is produced byintroducing a transgene into the germ line of the non-human animal.Transgenes can be introduced into embryonal target cells at variousdevelopmental stages. Different methods are used depending on the stageof development of the embryonal target cell. The specific line(s) of anyanimal used should, if possible, be selected for general good health,good embryo yields, good pronuclear visibility in the embryo, and goodreproductive fitness.

Introduction of the transgene into the embryo can be accomplished by anyof a variety of means known in the art such as microinjection,electroporation, lipofection or a viral vector. For example, thetransgene can be introduced into a mammal by microinjection of theconstruct into the pronuclei of the fertilized mammalian egg(s) to causeone or more copies of the construct to be retained in the cells of thedeveloping mammal(s). Following introduction of the transgenic constructinto the fertilized egg, the egg can be incubated in vitro for varyingamounts of time, or reimplanted into the surrogate host, or both. Onecommon method is to incubate the embryos in vitro for about 1-7 days,depending on the species, and then reimplant them into the surrogatehost.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the construct (e.g., by Southern blot analysis) of asegment of tissue. An embryo having one or more copies of the exogenouscloned construct stably integrated into the genome can be used toestablish a permanent transgenic animal line carrying the transgenicallyadded construct.

Transgenically altered animals can be assayed after birth for theincorporation of the construct into the genome of the offspring. Thiscan be done by hybridizing a probe corresponding to the DNA sequencecoding for the polypeptide or a segment thereof onto chromosomalmaterial from the progeny. Those progeny found to contain at least onecopy of the construct in their genome are grown to maturity.

Methods of producing transgenic avians are also known in the art, see,e.g., U.S. Pat. No. 5,162,215.

Nicotinamide riboside-related test agents can be obtained from a widevariety of sources including libraries of synthetic or naturalcompounds. Such agents can include analogs or derivatives ofnicotinamide riboside as well as tiazofurin and benzamide riboside andanalogs or derivatives thereof.

Alternatively, the isolated Nrk polypeptide can be used to generate acrystal structure of Nrk and synthetic nicotinamide riboside analogs canbe designed. Based on the crystal structure of E. coli panK, Asp127appears to play a key role in transition-state stabilization of thetransferring phosphoryl group of a pantothenate kinase (Yun, et al.(2000) J. Biol. Chem. 275:28093-28099). Accordingly, it is contemplatedthe corresponding Nrk mutant, e.g., NRK2-E100Q, can be used to generatea stable complex between an Nrk and a nucleotides (i.e.,Nrk2-E100Q+nicotinamide riboside+ATP can be stable enough tocrystallize). Alternatively, Nrk can produce a stable complex in thepresence of an inhibitor such as an ATP-mimetic compound (e.g., AMP-PNHPand AMP-PCH₂P). For metabolite kinases, bisubstrate inhibitors have beenvery successfully employed. For example, thymidylate kinase, whichperforms the reaction, dTMP+ATP->dTDP+AMP, is strongly inhibited bydTpppppA (Bone, et al. (1986) J. Biol. Chem. 261:16410-16413) andcrystal structures were obtained with this inhibitor (Lavie, et al.(1998) Biochemistry 37:3677-3686).

It has been shown that the best inhibitors typically contain one or twomore phosphates than the two substrates combined (i.e., dTppppA is notas good a substrate as dTpppppA). On the basis of the same types ofresults with adenosine kinase (Bone, et al. (1986) supra), it iscontemplated that NrppppA (i.e., an NAD+ analog with two extraphosphates) will be a better inhibitor than NrpppA (i.e., an NAD+ analogwith an extra phosphate, or, indeed, nicotinamide riboside+AppNHp). NAD+analogs with extra phosphates can be generated using standard enzymaticmethods (see, e.g., Guranowski, et al. (1990) FEBS Lett. 271:215-218)optimized for making a wide variety of adenylylated dinucleosidepolyphosphates (Fraga, et al. (2003) FEBS Lett. 543:37-41), namelyreaction of Nrpp (nicotinamide riboside diphosphate) and Nrppp(nicotinamide riboside triphosphate) with firefly luciferase-AMP. Thediphosphorylated form of NMN (Nrpp) is prepared with either uridylatekinase or cytidylate kinase (NMN+ATP->Nrpp). The triphosphorylated formof NMN (Nrppp) is subsequently prepared with nucleoside diphosphatekinase (Nrpp+ATP->Nrppp). The resulting inhibitors are then used incrystallization trials and/or are soaked into Nrk crystals.

Once the three-dimensional structure of Nrk is determined, a potentialtest agent can be examined through the use of computer modeling using adocking program such as GRAM, DOCK, or AUTODOCK (Dunbrack, et al. (1997)Folding & Design 2:27-42). This procedure can include computer fittingof potential agents to Nrk to ascertain how well the shape and thechemical structure of the potential ligand will interact with Nrk.Computer programs can also be employed to estimate the attraction,repulsion, and steric hindrance of the test agent. Generally the tighterthe fit (e.g., the lower the steric hindrance, and/or the greater theattractive force) the better substrate the agent will be since theseproperties are consistent with a tighter binding constraint.Furthermore, the more specificity in the design of a potential testagent the more likely that the agent will not interfere with relatedmammalian proteins. This will minimize potential side-effects due tounwanted interactions with other proteins.

The invention is also a method of treating cancer in a patient, havingor suspected of having cancer, with an isolated nucleic acid, deliveryvector, or polypeptide of the invention in combination with anicotinamide riboside-related prodrug. Administration of the nucleicacid, delivery vector, or polypeptide of the present invention to ahuman subject or an animal can be by any means known in the art foradministering nucleic acids, vectors, or polypeptides. A patient, asused herein, is intended to include any mammal such as a human,agriculturally-important animal, pet or zoological animal. A patienthaving or suspected of having a cancer is a patient who exhibits signsor symptoms of a cancer or because of inheritance, environmental ornatural reasons is suspected of having cancer. Nucleic acids encodingNrk, vectors containing the same, or Nrk polypeptides can beadministered to the subject in an amount effective to decrease,alleviate or eliminate the signs or symptoms of a cancer (e.g., tumorsize, feelings of weakness, and pain perception). The amount of theagent required to achieve the desired outcome of decreasing, eliminatingor alleviating a sign or symptom of a cancer will be dependent on thepharmaceutical composition of the agent, the patient and the conditionof the patient, the mode of administration, the type of condition ordisease being prevented or treated, age and species of the patient, theparticular vector, and the nucleic acid to be delivered, and can bedetermined in a routine manner.

While the prodrug and the Nrk nucleic acid, delivery vector, orpolypeptide can be delivered concomitantly, in an alternative embodimentthe Nrk nucleic acid, delivery vector, or polypeptide is provided first,followed by administration of the prodrug to precondition the cells togenerate the activated or toxic drug.

Types of cancers which can be treated in accordance with the method ofthe invention include, but are not limited to, pancreatic cancer,endometrial cancer, small cell and non-small cell cancer of the lung(including squamous, adneocarcinoma and large cell types), squamous cellcancer of the head and neck, bladder, ovarian, cervical, breast, renal,CNS, and colon cancers, myeloid and lymphocyltic leukemia, lymphoma,hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma,retinoblastoma, and sarcomas of the soft tissue and bone.

Typically, with respect to viral vectors, at least about 10³ virusparticles, at least about 10⁵ virus particles, at least about 10⁷ virusparticles, at least about 10⁹ virus particles, at least about 10¹¹ virusparticles, at least about 10¹² virus particles, or at least about 10¹³virus particles are administered to the patient per treatment. Exemplarydoses are virus titers of about 10⁷ to about 10¹⁵ particles, about 10⁷to about 10¹⁴ particles, about 10⁸ to about 10¹³ particles, about 10¹⁰to about 10¹⁵ particles, about 10¹¹ to about 10¹⁵ particles, about 10¹²to about 10¹⁴ particles, or about 10¹² to about 10¹³ particles.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve therapeutic levels of nucleic acid expression.

Tiazofurin is a nucleoside analog initially synthesized to be a cytidinedeaminase inhibitor. Tiazofurin was shown to be a prodrug that isconverted by cellular enzymes to TAD, an analog of NAD+, that inhibitsIMP dehydrogenase, the rate limiting enzyme in producing GTP and dGTP(Cooney, et al. (1983) supra). In phase I/II trials of acute leukemia,tiazofurin produced response rates as high as 85% and was granted orphandrug status for treatment of CML in accelerated phase or blast crisis.Treatment of cultured cells has shown that tiazofurin selectively killscancer cells by induction of apoptosis: the activity has been attributedboth to the increased dependence of actively replicating cells on dGTPand to the addiction of many transformed genotypes to signaling throughlow molecular weight G proteins (Jayaram, et al. (2002) Curr. Med. Chem.9:787-792). Examination of the sensitivity of the NCl-60 panel of cancercell lines and the literature on tiazofurin indicates that particularbreast, renal, CNS, colon and non-small cell lung-derived tumors areamong the most sensitive while others from the same organ sites areamong the most resistant (Johnson, et al. (2001) Br. J. Cancer84:1424-1431). As was demonstrated herein, the function of nicotinamideriboside as an NAD+ precursor is entirely dependent on Nrk1 and humanNrks have at least as high specific activity in tiazofurinphosphorylation as in nicotinamide riboside phosphorylation. BecauseNrk2 expression is muscle-specific (Li, et al. (1999) supra), and Nrk1is expressed at a very low level (Boon, et al. (2002) supra), whileNMN/NaMNAT is not restricted, it is contemplated that stratification oftumors by Nrk gene expression will largely predict and account fortiazofurin sensitivity.

Accordingly, the present invention is further a method for identifyingan individual or tumor which is susceptible to treatment with anicotinamide riboside-related prodrug. In one embodiment, the level ofNrk protein in an individual or tumor is detected by binding of aNrk-specific antibody in an immunoassay. In another embodiment, thelevel of Nrk enzyme activity is determined using, for example, thenicotinamide riboside phosphorylation assay disclosed herein. In anotherembodiment, the level of Nrk RNA transcript is determined using anynumber of well-known RNA-based assays for detecting levels of RNA. Oncedetected, the levels of Nrk are compared to a known standard. A changein the level of Nrk, as compared to the standard, is indicative of analtered level of susceptibility to treatment with a nicotinamideriboside-related prodrug. In a still further embodiment, mutations orpolymorphisms in the Nrk gene can be identified which result in analtered level of susceptibility to treatment with a nicotinamideriboside-related prodrug.

Optimized treatments for cancer and other diseases with nicotinamideriboside-related prodrugs are directed toward cells with naturally highlevels of an Nrk provided herein or toward cells which have beenrecombinantly engineered to express elevated levels of an Nrk. Safety,specificity and efficacy of these treatments can be modulated bysupplementation with or restriction of the amounts of any of the NAD+precursors, namely tryptophan, nicotinic acid, nicotinamide, ornicotinamide riboside.

For the detection of Nrk protein levels, antibodies which specificallyrecognize Nrk are generated. These antibodies can be either polyclonalor monoclonal. Moreover, such antibodies can be natural or partially orwholly synthetically produced. All fragments or derivatives thereof(e.g., Fab, Fab′, F(ab′)₂, scFv, Fv, or Fd fragments) which maintain theability to specifically bind to and recognize Nrk are also included. Theantibodies can be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE.

The Nrk-specific antibodies can be generated using classical cloning andcell fusion techniques. See, for example, Kohler and Milstein (1975)Nature 256:495-497; Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. Alternatively,antibodies which specifically bind Nrk are derived by a phage displaymethod. Methods of producing phage display antibodies are well-known inthe art (e.g., Huse, et al. (1989) Science 246(4935):1275-81).

Selection of Nrk-specific antibodies is based on binding affinity andcan be determined by various well-known immunoassays including,enzyme-linked immunosorbent, immunodiffusion chemiluminescent,immunofluorescent, immunohistochemical, radioimmunoassay, agglutination,complement fixation, immunoelectrophoresis, and immunoprecipitationassays and the like which can be performed in vitro, in vivo or in situ.Such standard techniques are well-known to those of skill in the art(see, e.g., “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi,eds. John Wiley & Sons, 1980; Campbell et al., “Methods and Immunology”,W. A. Benjamin, Inc., 1964; and Oellerich, M. (1984) J. Clin. Chem.Clin. Biochem. 22:895-904).

Once fully characterized for specificity, the antibodies can be used indiagnostic or predictive methods to evaluate the levels of Nrk inhealthy and diseased tissues (i.e., tumors) via techniques such asELISA, western blotting, or immunohistochemistry.

The general method for detecting levels of Nrk protein providescontacting a sample with an antibody which specifically binds Nrk,washing the sample to remove non-specific interactions, and detectingthe antibody-antigen complex using any one of the immunoassays describedabove as well a number of well-known immunoassays used to detect and/orquantitate antigens (see, for example, Harlow and Lane (1988) supra).Such well-known immunoassays include antibody capture assays, antigencapture assays, and two-antibody sandwich assays.

For the detection of nucleic acid sequences encoding Nrk, either aDNA-based or RNA-based method can be employed. DNA-based methods fordetecting mutations in an Nrk locus (i.e., frameshift mutations, pointmutations, missense mutations, nonsense mutations, splice mutations,deletions or insertions of induced, natural or inherited origin)include, but are not limited to, DNA microarray technologies,oligonucleotide hybridization (mutant and wild-type), PCR-basedsequencing, single-strand conformational polymorphism (SSCP) analysis,heteroduplex analysis (HET), PCR, or denaturing gradient gelelectrophoresis. Mutations can appear, for example, as a dual base callon sequencing chromatograms. Potential mutations are confirmed bymultiple, independent PCR reactions. Exemplary single nucleotidepolymorphisms which can be identified in accordance with the diagnosticmethod of the invention include, but are not limited to, NCBI SNPCluster ID Nos. rs3752955, rs1045882, rs11519, and rs3185880 for humanNrk1 and Cluster ID Nos. rs2304190, rs4807536, and rs1055767 for humanNrk2.

To detect the levels of RNA transcript encoding the Nrk, nucleic acidsare isolated from cells of the individual or tumor, according tostandard methodologies (e.g., Sambrook et al. (1989) Molecular Cloning,a Laboratory Manual, Cold Spring Harbor Laboratories, New York). Thenucleic acid can be whole cell RNA or fractionated to Poly-A+. It may bedesirable to convert the RNA to a complementary DNA (cDNA). Normally,the nucleic acid is amplified.

A variety of methods can be used to evaluate or quantitate the level ofNrk RNA transcript present in the nucleic acids isolated from anindividual or tumor. For example, levels of Nrk RNA transcript can beevaluated using well-known methods such as northern blot analysis (see,e.g., Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual,Cold Spring Harbor Laboratories, New York); oligonucleotide or cDNAfragment hybridization wherein the oligonucleotide or cDNA is configuredin an array on a chip or wafer; real-time PCR analysis, or RT-PCRanalysis.

Suitable primers, probes, or oligonucleotides useful for such detectionmethods can be generated by the skilled artisan from the Nrk nucleicacid sequences provided herein. The term primer, as defined herein, ismeant to encompass any nucleic acid that is capable of priming thesynthesis of a nascent nucleic acid in a template-dependent process.Typically, primers are oligonucleotides from ten to twenty base pairs inlength, but longer sequences can be employed. Primers can be provided indouble-stranded or single-stranded form. Probes are defined differently,although they can act as primers. Probes, while perhaps capable ofpriming, are designed for binding to the target DNA or RNA and need notbe used in an amplification process. In one embodiment, the probes orprimers are labeled with, for example, radioactive species (³²P, ¹⁴C,³⁵S, ³H, or other label) or a fluorophore (rhodamine, fluorescein).Depending on the application, the probes or primers can be used cold,i.e., unlabeled, and the RNA or cDNA molecules are labeled.

Depending on the format, detection can be performed by visual means(e.g., ethidium bromide staining of a gel). Alternatively, the detectioncan involve indirect identification of the product viachemiluminescence, radiolabel or fluorescent label or even via a systemusing electrical or thermal impulse signals (Bellus (1994) J. Macromol.Sci. Pure Appl. Chem. A311:1355-1376).

After detecting mutations in Nrk or the levels of Nrk present in anindividual or tumor, said mutations or levels are compared with a knowncontrol or standard. A known control can be a statistically significantreference group of individuals that are susceptible or lacksusceptibility to treatment with a nicotinamide riboside-related prodrugto provide diagnostic or predictive information pertaining to theindividual or tumor upon which the analysis was conducted.

As described herein, nicotinamide riboside isolated from deproteinizedwhey fraction of cow's milk was sufficient to support NRK1-dependentgrowth in a qns1 mutant. Accordingly, mutant strains generated hereinwill be useful in identifying other natural or synthetic sources fornicotinamide riboside for use in dietary supplements. Thus, the presentinvention also encompasses is a method for identifying such natural orsynthetic sources. As a first step of the method, a first cell lacking afunctional glutamine-dependent NAD+ synthetase is contacted with anisolated extract from a natural or synthetic source. In one embodiment,the first cell is a qns1 mutant (i.e., having no NAD+ synthetase)carrying the QNS1 gene on a URA3 plasmid. While any cell can be used, inparticular embodiments a yeast cell is used in this method of theinvention. A qns1 mutant strain has normal growth on 5-fluoroorotic acid(i.e., cured of the URA3 QNS1 plasmid) as long as it is supplied withnicotinamide riboside.

As a second step of the method, a second cell lacking a functionalglutamine-dependent NAD+ synthetase and a functional nicotinamideriboside kinase is contacted with the same isolated extract from thenatural or synthetic source of the prior step. Using a qns1 and nrk1double mutant, it was demonstrated herein that the NRK1 gene isnecessary for growth on nicotinamide riboside: qns1 and nrk1 aresynthetically lethal even with nicotinamide riboside. This deletionstrain is useful in this screening assay of the invention as it allowsone to distinguish between nicotinamide riboside, NMN and NAD+ as theeffective nutrient.

As a subsequent step of the method, the growth of the first cell andsecond cell are compared. If the isolated extract contains anicotinamide riboside, the first cell will grow and the second cell willnot.

Synthetic sources of nicotinamide riboside can include any library ofchemicals commercially available from most large chemical companiesincluding Merck, Glaxo, Bristol Meyers Squibb, Monsanto/Searle, EliLilly and Pharmacia. Natural sources which can be tested for thepresence of a nicotinamide riboside include, but are not limited to,cow's milk, serum, meats, eggs, fruit and cereals. Isolated extracts ofthe natural sources can be prepared using standard methods. For example,the natural source can be ground or homogenized in a buffered solution,centrifuged to remove cellular debris, and fractionated to remove salts,carbohydrates, polypeptides, nucleic acids, fats and the like beforebeing tested on the mutants strains of the invention. Any source ofnicotinamide riboside that scores positively in the assay of theinvention can be further fractionated and confirmed by standard methodsof HPLC and mass spectrometry.

Nicotinic acid is an effective agent in controlling low-densitylipoprotein cholesterol, increasing high-density lipoproteincholesterol, and reducing triglyceride and lipoprotein (a) levels inhumans (see, e.g., Miller (2003) Mayo Clin. Proc. 78(6):735-42). Thoughnicotinic acid treatment effects all of the key lipids in the desirabledirection and has been shown to reduce mortality in target populations,its use is limited because of a side effect of heat and redness termedflushing, which is significantly effected by the nature of formulation.Further, nicotinamide protects against stroke injury in model systems,due to multiple mechanisms including increasing mitochondrial NAD+levels and inhibiting PARP (Klaidman, et al. (2003) Pharmacology69(3):150-7). Altered levels of NAD+ precursors have been shown toeffect the regulation of a number of genes and lifespan in yeast(Anderson, et al. (2003) Nature 423(6936):181-5).

NAD+ administration and NMN adenylyltransferase (Nmnat1) expression havealso been shown to protect neurons from axonal degeneration (Araki, etal. (2004) Science 305:1010-1013). Because nicotinamide riboside is asoluble, transportable nucleoside precursor of NAD+, nicotinamideriboside can be used to protect against axonopathies such as those thatoccur in Alzheimer's Disease, Parkinson's Disease and MultipleSclerosis. Expression of the NRK1 or NRK2 genes, or directadministration of nicotinamide riboside or a stable nicotinamideriboside prodrug, could also protect against axonal degeneration.

NMN adenylytransferase overexpression has been shown to protect neuronsfrom the axonopathies that develop with ischemia and toxin exposure,including vincristine treatment (Araki, et al. (2004) Science305:1010-1013). Vincristine is one of many chemotherapeutic agents whoseuse is limited by neurotoxicity. Thus, administration of nicotinamideriboside or an effective nicotinamide riboside prodrug derivative couldbe used to protect against neurotoxicity before, during or aftercytotoxic chemotherapy.

Further, conversion of benign Candida glabrata to the adhesive,infective form is dependent upon the expression of EPA genes encodingadhesins whose expression is mediated by NAD+ limitation, which leads todefective Sir2-dependent silencing of these genes (Domergue, et al.(March 2005) Science, 10.1126/science.1108640). Treatment with nicotinicacid reduces expression of adhesins and increasing nicotinic acid inmouse chow reduces urinary tract infection by Candida glabrata. Thus,nicotinamide riboside can be used in the treatment of fungal infections,in particular, those of Candida species by preventing expression ofadhesins.

Accordingly, agents (e.g., nicotinamide riboside) that work through thediscovered nicotinamide riboside kinase pathway of NAD+ biosynthesiscould have therapeutic value in improving plasma lipid profiles,preventing stroke, providing neuroprotection with chemotherapytreatment, treating fungal infections, preventing or reducingneurodegeneration, or in prolonging health and well-being. Thus, thepresent invention is further a method for preventing or treating adisease or condition associated with the nicotinamide riboside kinasepathway of NAD+ biosynthesis by administering an effective amount of anicotinamide riboside composition. Diseases or conditions whichtypically have altered levels of NAD+ or NAD+ precursors or couldbenefit from increased NAD+ biosynthesis by treatment with nicotinamideriboside include, but are not limited to, lipid disorders (e.g.,dyslipidemia, hypercholesterolaemia or hyperlipidemia), stroke,neurodegenerative diseases (e.g., Alzheimer's, Parkinsons and MultipleSclerosis), neurotoxicity as observed with chemotherapies, Candidaglabrata infection, and the general health declines associated withaging. Such diseases and conditions can be prevented or treated bysupplementing a diet or a therapeutic treatment regime with anicotinamide riboside composition.

The source of nicotinamide riboside can be from a natural or syntheticsource identified by the method of the instant invention, or can bechemically synthesized using established methods (Tanimori (2002)Bioorg. Med. Chem. Lett. 12:1135-1137; Franchetti (2004) Bioorg. Med.Chem. Lett. 14:4655-4658). In addition, the nicotinamide riboside can bea derivative (e.g., L-valine or L-phenylalanine esters) of nicotinamideriboside. For example, an L-valyl (valine) ester on the 5′ O ofacyclovir (valacyclovir) improved the pharmacokinetic properties of thedrug by promoting transport and allowing cellular delivery of thenucleoside after hydrolysis by an abundant butyryl esterase (Han, et al.(1998) Pharm. Res. 15:1382-1386; Kim, et al. (2003) J. Biol. Chem.278:25348-25356). Accordingly, the present invention also encompassesderivatives of nicotinamide riboside, in particular L-valine orL-phenylalanine esters of nicotinamide riboside, which are contemplatedas having improved pharmacokinetic properties (e.g., transport anddelivery). Such derivatives can be used alone or formulated with apharmaceutically acceptable carrier as disclosed herein.

An effective amount of nicotinamide riboside is one which prevents,reduces, alleviates or eliminates the signs or symptoms of the diseaseor condition being prevented or treated and will vary with the diseaseor condition. Such signs or symptoms can be evaluated by the skilledclinician before and after treatment with the nicotinamide riboside toevaluate the effectiveness of the treatment regime and dosages can beadjusted accordingly.

As alterations of NAD+ metabolism may need to be optimized forparticular conditions, it is contemplated that nicotinamide ribosidetreatments can further be used in combination with other NAD+precursors, e.g., tryptophan, nicotinic acid and/or nicotinamide.

Polypeptides, nucleic acids, vectors, dietary supplements (i.e.nicotinamide riboside), and nicotinamide riboside-related prodrugsproduced or identified in accordance with the methods of the inventioncan be conveniently used or administered in a composition containing theactive agent in combination with a pharmaceutically acceptable carrier.Such compositions can be prepared by methods and contain carriers whichare well-known in the art. A generally recognized compendium of suchmethods and ingredients is Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippingcott Williams &Wilkins: Philadelphia, Pa., 2000. A carrier, pharmaceutically acceptablecarrier, or vehicle, such as a liquid or solid filler, diluent,excipient, or solvent encapsulating material, is involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must beacceptable in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient.

Examples of materials which can serve as carriers include sugars, suchas lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in formulations. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Polypeptides, nucleic acids, vectors, dietary supplements, andnicotinamide riboside-related prodrugs produced or identified inaccordance with the methods of the invention, hereafter referred to ascompounds, can be administered via any route include, but not limitedto, oral, rectal, topical, buccal (e.g., sub-lingual), vaginal,parenteral (e.g., subcutaneous, intramuscular including skeletal muscle,cardiac muscle, diaphragm muscle and smooth muscle, intradermal,intravenous, intraperitoneal), topical (i.e., both skin and mucosalsurfaces, including airway surfaces), intranasal, transdermal,intraarticular, intrathecal and inhalation administration,administration to the liver by intraportal delivery, as well as directorgan injection (e.g., into the liver, into the brain for delivery tothe central nervous system). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular compound which is being used.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor (BASF, Parsippany, N.J.). For othermethods of administration, the carrier can be either solid or liquid.

For oral therapeutic administration, the compound can be combined withone or more carriers and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums, foods and the like. Such compositions and preparationsshould contain at least 0.1% of active compound. The percentage of thecompound and preparations can, of course, be varied and can convenientlybe between about 0.1 to about 100% of the weight of a given unit dosageform. The amount of active compound in such compositions is such that aneffective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. The above listingis merely representative and one skilled in the art could envision otherbinders, excipients, sweetening agents and the like. When the unitdosage form is a capsule, it can contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials can be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules can be coated with gelatin,wax, shellac or sugar and the like.

A syrup or elixir can contain the active agent, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be substantially non-toxic inthe amounts employed. In addition, the active compounds can beincorporated into sustained-release preparations and devices including,but not limited to, those relying on osmotic pressures to obtain adesired release profile.

Formulations of the present invention suitable for parenteraladministration contain sterile aqueous and non-aqueous injectionsolutions of the compound, which preparations are generally isotonicwith the blood of the intended recipient. These preparations can containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include suspending agents andthickening agents. The formulations can be presented in unit\dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

Formulations suitable for topical application to the skin can take theform of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.Carriers which can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the compound. Suitable formulations contain citrate orbis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2 M ofthe compound.

A compound can alternatively be formulated for nasal administration orotherwise administered to the lungs of a subject by any suitable means.In particular embodiments, the compound is administered by an aerosolsuspension of respirable particles containing the compound, which thesubject inhales. The respirable particles can be liquid or solid. Theterm aerosol includes any gas-borne suspended phase, which is capable ofbeing inhaled into the bronchioles or nasal passages. Specifically,aerosol includes a gas-borne suspension of droplets, as can be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition suspended in air or other carriergas, which can be delivered by insufflation from an inhaler device, forexample. See Ganderton & Jones, Drug Delivery to the Respiratory Tract,Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic DrugCarrier Systems 6:273-313; and Raeburn, et al. (1992) J. Pharmacol.Toxicol. Methods 27:143-159. Aerosols of liquid particles containing thecompound can be produced by any suitable means, such as with apressure-driven aerosol nebulizer or an ultrasonic nebulizer, as isknown to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.Aerosols of solid particles containing the compound can likewise beproduced with any solid particulate medicament aerosol generator, bytechniques known in the pharmaceutical art.

Alternatively, one can administer the compound in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well-known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same can be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed can be of anyconventional composition and can either contain cholesterol or can becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced can be reduced insize, as through the use of standard sonication and homogenizationtechniques.

A liposomal formulation containing a compound disclosed herein or saltthereof, can be lyophilized to produce a lyophilizate which can bereconstituted with a carrier, such as water, to regenerate a liposomalsuspension.

In particular embodiments, the compound is administered to the subjectin an effective amount, as that term is defined herein. Dosages ofactive compounds can be determined by methods known in the art, see,e.g., Remington: The Science and Practice of Pharmacy, Alfonso R.Gennaro, editor, 20th ed. Lippingcott Williams & Wilkins: Philadelphia,Pa., 2000. The selected effective dosage level will depend upon avariety of factors including the activity of the particular compound ofthe present invention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell-known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required for prevention or treatment in an animal subjectsuch as a human, agriculturally-important animal, pet or zoologicalanimal.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 S. cerevisiae Strains

Yeast diploid strain BY165, heterozygous for qns1 deletion and haploidBY165-1d carrying a chromosomal deletion of qns1 gene, transformed withplasmid pB175 containing QNS1 and URA3 is known in the art (Bieganowski,et al. (2003) supra). Genetic deletions were introduced by directtransformation with PCR products (Brachmann, et al. (1998) Yeast14:115-132) generated from primers. After 24 hours of growth on completemedia, cells were plated on media containing 5-fluoroorotic acid (Boeke,et al. (1987) Methods Enzymol. 154:164-175). The ado1 disruptioncassette was constructed by PCR with primers 7041 (5′-CTA TTT AGA GTAAGG ATA TTT TTT CGG AAG GGT AAG AGG GAC CAA CTT CTT CTG TGC GGT ATT TCACAC CG-3′; SEQ ID NO:10) and 7044 (5′-ATG ACC GCA CCA TTG GTA GTA TTGGGT AAC CCA CTT TTA GAT TTC CAA GCA GAT TGT ACT GAG AGT GCA C-3′; SEQ IDNO:11) and plasmid pRS413 as a template. Yeast strain BY165 wastransformed with this PCR product, and homologous recombination inhistidine prototrophic transformants was confirmed by PCR with primers7042 (5′-AAG CTA GAG GGA ACA CGT AGA G-3′; SEQ ID NO:12) and 7043(5′-TTA TCT TGT GCA GGG TAG AAC C-3′; SEQ ID NO:13). This strain wastransformed with plasmid pB175 and subjected to sporulation and tetraddissection. Haploid strain BY237, carrying qns1 and ado1 deletions andplasmid, was selected for further experiments. The urk1 deletion wasintroduced into strain BY237 by transformation with the product of thePCR amplification that used pRS415 as a template and PCR primers 7051(5′-CGA TCT TCA TCA TTT ATT TCA ATT TTA GAC GAT GAA ACA AGA GAC ACA TTAGAT TGT ACT GAG AGT GCA C-3′; SEQ ID NO:14) and 7052 (5′-AAA ATA CTT TGAATC AAA AAA TCT GGT CAA TGC CCA TTT GTA TTG ATG ATC TGT GCG GTA TTT CACACC G-3′; SEQ ID NO:15). Disruption was confirmed by PCR with primers7053 (5′-ATG TCC CAT CGT ATA GCA CCT TCC-3′; SEQ ID NO:16) and 7054(5′-GCC TCT AAT TAT TCT CAA TCA CAA CC-3′; SEQ ID NO:17), and theresulting strain was designated BY247. The rbk1 disruption cassette wasconstructed by PCR with primers 7063 (5′-AAA CTT TCA GGG CTA ACC ACT TCGAAA CAC ATG CTG GTG GTA AGG GAT TGA GAT TGT ACT GAG AGT GCA C-3′; SEQ IDNO:18) and 7065 (5′-GAA CAG AAA AGC ACC CCT CTC GAA CCC AAA GTC ATA ACCACA ATT CCT CTC TGT GCG GTA TTT CAC ACC G-3′; SEQ ID NO:19) and plasmidpRS411 as a template. Disruption was introduced into strain BY242 bytransformation with the product of this reaction and confirmed by PCRwith primers 7062 (5′-GGA TAG ATT ACC TAA CGC TGG AG-3′; SEQ ID NO:20)and 7064 (5′-TTG TAC TTC AGG GCT TTC GTG C-3′; SEQ ID NO:21). Theresulting strain, carrying deletions of qns1, ado1, urk1 and rbk1 geneswas designated BY252. A yeast strain carrying disruption of the NRK1locus was made by transformation of the strain BY165-1d with the HIS3marker introduced into disruption cassette by PCR with primers 4750(5′-AAT AGC GTG CAA AAG CTA TCG AAG TGT GAG CTA GAG TAG AAC CTC AAA ATAGAT TGT ACT GAG AGT GCA C-3′; SEQ ID NO:22) and 4751 (5′-CTA ATC CTT ACAAAG CTT TAG AAT CTC TTG GCA CAC CCA GCT TAA AGG TCT GTG CGG TAT TTC ACACCG-3′; SEQ ID NO:23). Correct integration of the HIS3 marker into NRK1locus was confirmed by PCR with primers 4752 (5′-ACC AAC TTG CAT TTT AGGCTG TTC-3′; SEQ ID NO:24) and 4753 (5′-TAA GTT ATC TAT CGA GGT ACA CATTC-3′; SEQ ID NO:25).

EXAMPLE 2 Nicotinamide Riboside and Whey Preparations

NMN (39.9 mg; Sigma, St. Louis, Mo.) was treated with 1250 units of calfintestinal alkaline phosphatase (Sigma) for 1 hour at 37° C. in 1 mL 100mM NaCl, 20 mM Tris pH 8.0, 5 mM MgCl₂. Hydrolysis of NMN tonicotinamide riboside was verified by HPLC and phosphatase was removedby centrifuging the reaction through a 5,000 Da filter (Millipore,Billerica, Mass.). A whey vitamin fraction of commercial nonfat cow'smilk was prepared by adjusting the pH to 4 with HCl, stirring at 55° C.for 10 minutes, removal of denatured casein by centrifugation, andpassage through a 5,000 Da filter. In yeast media, nicotinamide ribosidewas used at 10 μM and whey vitamin fraction at 50% by volume.

EXAMPLE 3 Yeast GST-ORF Library

Preparation of the fusion protein library was in accordance withwell-established methods (Martzen, et al. (1999) supra; Phizicky, et al.(2002) Methods Enzymol. 350:546-559) at a 0.5 liter culture scale foreach of the 64 pools of 90-96 protein constructs. Ten percent of eachpool preparation was assayed for Nrk activity in overnight incubations.

EXAMPLE 4 Nicotinamide Riboside Phosphorylation Assays

Reactions (0.2 mL) containing 100 mM NaCl, 20 mM NaHEPES pH 7.2, 5 mMβ-mercaptoethanol, 1 mM ATP, 5 mM MgCl₂, and 500 μM nicotinamideriboside or alternate nucleoside, were incubated at 30° C. andterminated by addition of EDTA to 20 mM and heating for 2 minutes at100° C. Specific activity assays, containing 50 ng to 6 μg enzymedepending on the enzyme and substrate, were incubated for 30 minutes at30° C. to maintain initial rate conditions. Reaction products wereanalyzed by HPLC on a strong anion exchange column with a 10 mM to 750mM gradient of KPO₄ pH 2.6.

EXAMPLE 5 NRK Gene and cDNA Cloning and Enzyme Purification

The S. cerevisiae NRK1 gene was amplified from total yeast DNA withprimers 7448 (5′-CGC TGC ACA TAT GAC TTC GAA AAA AGT GAT ATT AGT TGC-3′;SEQ ID NO:26) and 7449 (5′-CCG TCT CGA GCT AAT CCT TAC AAA GCT TTA GAATCT CTT GG-3′; SEQ ID NO:27). The amplified DNA fragment was cloned invector pSGO4 (Ghosh and Lowenstein (1997) Gene 176:249-255) for E. coliexpression using restriction sites for NdeI and XhoI included in primersequences and the resulting plasmid was designated pB446. Samples ofcDNA made from human lymphocytes and spleen were used as a template foramplification of human NRK1 using primers 4754 (5′-CCG GCC CAT GGC GCACCA CCA TCA CCA CCA TCA TAT GAA AAC ATT TAT CAT TGG AAT CAG TGG-3′; SEQID NO:28) and 4755 (5′-GCG GGG ATC CTT ATG CTG TCA CTT GCA AAC ACT TTTGC-3′; SEQ ID NO:29). For E. coli expression, PCR amplicons from thisreaction were cloned into restriction sites NcoI and BamHI of vectorpMR103 (Munson, et al. (1994) Gene 144:59-62) resulting in plasmidpB449. Subsequently, plasmid pB449 was used as a template for PCR withprimers 7769 (5′-CCG CGG ATC CAT GAA AAC ATT TAT CAT TGG AAT CAG TGG-3′;SEQ ID NO:30) and 7770 (5′-GCC GCT CGA GTT ATG CTG TCA CTT GCA AAC ACTT-3′; SEQ ID NO:31). The product of this amplification was clonedbetween BamHI and XhoI sites of vector p425GAL1 (Mumberg, et al. (1994)Nucleic Acids Res. 22:5767-5768) and the resulting plasmid carryinghuman NRK1 gene under GAL1 promoter control was designated pB450. HumanNRK2 cDNA was amplified with primers 7777 (5′-GGC AGG CAT ATG AAG CTCATC GTG GGC ATC G-3′; SEQ ID NO:32) and 7776 (5′-GCT CGC TCG AGT CAC ATGCTG TCC TGC TGG GAC-3′; SEQ ID NO:33). The amplified fragment wasdigested with NdeI and XhoI enzymes and cloned in plasmid pSGA04 for E.coli expression. His-tagged enzymes were purified with Ni-NTA agarose.

1. A pharmaceutical composition comprising nicotinamide riboside inadmixture with a carrier, wherein said composition is formulated fororal administration.
 2. The pharmaceutical composition of claim 1,wherein the nicotinamide riboside is isolated from a natural orsynthetic source.
 3. The pharmaceutical composition of claim 1, whereinthe formulation comprises a tablet, troche, capsule, elixir, suspension,syrup, wafer, chewing gum, or food.
 4. The pharmaceutical composition ofclaim 1, further comprising one or more of tryptophan, nicotinic acid,or nicotinamide.
 5. The pharmaceutical composition of claim 1 whichincrease NAD+ biosynthesis upon oral administration.